1. Field of the Invention
The present invention relates generally to the field of cell biology. More particularly, it concerns cell culture systems for salivary gland cells and production and use of salivary tissue-specific extracellular matrices for growth and differentiation of cells.
2. Description of Related Art
Salivary gland hypofunction is usually associated with xerostomic medications, radiotherapy to head and neck regions, autoimmune diseases (e.g., Sjögren's syndrome), aging, and systemic diseases such as diabetes, mellitus and renal diseases (Napenas, et al., 2009), which usually leads to rampant and severe oral diseases with compromised quality of life. Unfortunately, adult salivary glands are highly differentiated and show little regenerative capacity in response to physical (e.g. radiation) and pathological (e.g. Sjögren's syndrome) assaults. Therefore, development of strategies to preserve or regain secretory components in the salivary gland is essential for the management of patients with salivary diseases. Development of these treatment strategies requires the establishment of a system capable of replicating the salivary gland cell “niche” to support the proliferation and differentiation of salivary gland progenitor cells. The potential approaches for restoring the function of salivary glands include 1) inserting genes into residual salivary acinar or ductal cells, 2) replacing the salivary gland with functional artificial tissue, and 3) regrowing the salivary gland tissue in situ (Baum, 2000). The former can be achieved by gene transfer, but the latter 2 approaches will require extensive knowledge of stem cells and tissue engineering technologies. Reconstruction of salivary glands is a complex process that involves cell-cell communication, cell-matrix interaction and cell signal transduction in a 3-dimensional (3D) structure. To achieve these complex biological processes, several parallel lines of regeneration research have focused on identifying and/or isolating salivary stem/progenitor cells (Lombaert, et al., 2011; Kagami, et al., 2008), elucidating pathways and factors associated with salivary gland development (Harunaga, et al., 2011), and developing appropriate biomaterial scaffolds that support the proliferation and differentiation of salivary gland progenitors (Aframian & Palmon, 2008; Chan, et al., 2012).
Extracellular matrix (ECM) is an important component of the cellular niche in tissues, supplying critical biochemical and physical signals to initiate or sustain cellular functions (Chen, et al., 2008; Lai, et al., 2010). With advances in tissue engineering, the various scaffold biomaterials have been developed to mimic ECMs for tissue regeneration or repair (Nagaoka, et al., 2010). Among them, the materials that have been use to support the proliferation and differentiation of salivary gland progenitors include chitosan, polyglycolic acid (PGA), poly-(1)-lactic acid (PLLA), poly (lactic-co-glycolic acid) (PLAG), poly(ethylene glycol)-terephthalate (PEFT/poly (butylene terephthalate (PBT) (Kagami, et al., 2008; Chan, et al., 2012; Chen, et al., 2005). However, these polymeric scaffolds can induce inflammation resulting from the acidity of their degradation products (Athanasiou, et al., 1996; Cancedda, et al., 2003). Another potential scaffold material, Matrigel, which contains basement membrane proteins secreted by EHS mouse sarcoma cells, has been used to grow primary salivary gland epithelial cells in culture (Maria, et al., 2011). Although varying levels of success have been achieved with this product, it is not consistent with the long term goal to reconstitute the salivary gland niche (tissue-specific ECM) on a scaffold for controlling stem cell fate. Natural scaffold materials, especially silk, are desirable due to their wide ranges of elasticity (allowing tissue-specific scaffold formation), pore sizes (allowing tissue specific nutrition and oxygen access), low bacterial adherence, biodegradable, and low toxicity and immunogenicity (Leal-Egana & Scheibel, 2010).
Adult salivary glands are known to contain progenitor and stem cells that can be directed to salivary tissue differentiation or other tissue types depending on the tissue-specific microenvironment or niche that is mainly made up of ECM proteins associated with growth factors (Coppes & Stokman, 2011). Currently, the 3-D matrixes used for in vitro salivary cell growth and differentiation systems are either unpractical for clinical use, e.g., as tumor cell produced Matrigel, or only have one or two basement membrane components (Maria, et al., 2011). Basement membrane is critical for epithelial cell polarization and differentiation and has been demonstrated to play a key role during salivary gland development (Kadova & Yamashina, 2005). A tissue-specific ECM microenvironment is essential to provide chemical and physical cues to direct/govern multipotent stem cells in vivo and in vitro for tissue regeneration and repair (Chen, 2010; Costa, et al., 2012).
There remains a need for a tissue culture system to allow growth of salivary gland cells in such a way that they retain physiologically relevant features of salivary gland cell function. Also desirable are salivary gland tissue-specific three-dimensional (3D) scaffolds for salivary gland tissue engineering. Salivary gland-specific extracellular matrices can be used to differentiate salivary gland cell progenitors, including pluripotent stem cells, into salivary gland cells and to grow salivary gland tissue that can be used in a variety of therapies.